The present invention relates to a method for the quantitative determination of individual polyamine components, i.e. free polyamines and acetylated polyamines, in an aqueous mixture or, more particularly, to a method for the quantitative determination of individual contents of polyamine components in an aqueous mixture containing at least two of the polyamines selected from the group consisting of free polyamines, e.g. spermine, spermidine and putrescine, and acetylated polyamines, e.g. acetylspermine, N.sup.1 -acetylspermidine, N.sup.8 -acetylspermidine and acetylputrescine, including at least one of the said acetylated polyamines by an enzymatic means.
As is well known, polyamines or, in particular, free polyamines such as putrescine NH.sub.2 (CH.sub.2).sub.4 NH.sub.2, spermidine NH.sub.2 (CH.sub.2).sub.4 NH(CH.sub.2).sub.3 NH.sub.2 and spermine NH.sub.2 (CH.sub.2).sub.3 NH(CH.sub.2).sub.4 NH(CH.sub.2).sub.3 NH.sub.2 are widely found in almost all kinds of living bodies as a constituent of the cells not only in animals but also in plants and microorganisms and pertain to the division and multiplication of cells as well as the metabolism of nucleic acid as a biochemical background thereof.
It has been reported recently that the concentration of the polyamines in urine is larger from the patients of cancer than from healthy persons (Cancer Research, volume 31, pages 1555-1558) and investigations have been undertaken extensively by many investigators on the relationship between the contents of polyamines in the so-called body fluids such as urine, blood, limph and the like of a patient and the condition of cancer which the patient suffers. It is now understood that the polyamine metabolism is stimulated in the increasing period of the tumor tissues and fluctuation in the amount of polyamine excretion may be taken as a reflection of the transition of the condition of a patient suffering cancer so that it is eagerly desired to develop a rapid and convenient method for the quantitative determination of polyamine constituents in a sample taken from a patient of cancer as important information for the clinical diagnosis of cancer as well as for the therapeutic effect therefor.
Conventionally several methods have been undertaken for the purpose of quantitative determination of polyamines including a gas chromatographic method (see, for example, Clinical Chemistry, volume 19, pages 904-907), a method by use of an amino acid analyzer (see, for example, FEBS Letters, volume 46, pages 305-307) and a method of high-speed liquid chromatography (see, for example, Journal of Chromatography, volume 145, pages 141-146). These chemical methods constituting the major current of the analytical methods for polyamines are not quite satisfactory from the practical standpoint because these methods cannot be performed with sufficient rapidness and the procedures of these methods are complicated and lengthy due to the necessity of pretreatment to convert the polyamines into derivatives susceptible to the determination in the respective methods with different principles.
Alternatively, several enzymatic methods also have been proposed for the determination of polyamines by utilizing the specific activity of a particular enzyme including a method for the free polyamines (see, for example, Japanese Patent Publication No. 56-36918), a method for the determination of spermidine and spermine (see, for example, Japanese Patent Publication No. 56-21398) and a method for the determination of spermine, spermidine and putrescine (see, for example, Japanese Pat. Kokai No. 50-9492). These enzymatic methods are preferred to the above mentioned chemical methods because the troublesome pretreatment can be omitted for the conversion of the polyamines into the derivatives suitable for the determination.
According to the results of recent investigations, on the other hand, it is important in order to understand the condition of the patient of cancer to know not only the total amount of the polyamines but also the proportion of the respective free polyamines, i.e. spermidine, spermine and putrescine, as well as the proportion of the acetylated polyamines, for example, the proportion of acetylspermine, acetylspermidine and acetylputrescine or the proportion of N.sup.1 -acetylspermidine and N.sup.8 -acetylspermidine, while the above-mentioned enzymatic methods for the analysis are not suitable for the direct determination of the acetylated polyamines because the methods are for the free polyamines and the acetylated polyamines must be deacetylated to the corresponding free polyamines by the hydrolysis reaction with hydrochloric acid and the like or by an enzymatic method. Therefore, it has been eagerly desired to develop a rapid and convenient method for the quantitative determination of the individual polyamine components in a mixture containing not only free polyamines but also acetylated polyamines capable of giving very reliable results.